The human influenza virus exacts a fearsome toll on the economy and on public health (Majury, 2005; Falsey and Walsh, 2006). Despite vaccination programs and antiviral drugs, seasonal influenza alone causes an estimated 4000 Canadian deaths annually (Schanzer et al., 2007) and is the number one infectious cause of death in Ontario (Kuster et al., 2010). Influenza infects the respiratory epithelium and most deaths occur due to pulmonary complications. About 25% of deaths occur as a direct result of the initial viral infection (Louria et al., 1959), while the remainder are attributed to a superimposed bacterial infection (also called a bacterial superinfection), such as pneumonia from Staphylococcus aureus (Mohan et al., 2005). In both cases, respiratory deterioration is marked by acute lung injury (Dominguez-Cherit et al., 2009; Louria et al., 1959), a potentially fatal syndrome of pulmonary edema that occurs due to increased permeability of the lung microvasculature (Lee and Slutsky, 2001). Blood vessels in the lung are lined by a continuous layer of endothelium; thus, loss of barrier integrity of the lung microvascular endothelium is a prerequisite for acute lung injury. While antiviral drugs exist, they only partially reduce mortality (McGeer et al., 2007), they must be administered early to be effective, and their use is complicated by the rapid development of resistance. Thus, new therapies for the most severe cases of influenza are desperately needed.
Unlike high pathogenicity avian influenza viruses (e.g. H5N1 avian influenza) (Maines et al., 2008), human influenza strains lack certain basic amino acids in their hemagglutinin molecules; this limits cleavage to trypsin-like proteases that are contained within the respiratory tract. Thus human influenza primarily infects the respiratory epithelium leading to epithelial injury, apoptosis and desquamation (Kuiken and Taubenberger, 2008). In uncomplicated infections, these changes to the airway epithelium are transient and the process of repair is evident within days. However, in primary viral pneumonia, the virus also infects the distal lung, particularly type I pneumocytes and ciliated bronchiolar epithelium, leading to damage to the alveoli including frank alveolar denudement (Kuiken and Taubenberger, 2008); type II pneumocytes and alveolar macrophages can also be infected. In the 1957 flu pandemic, primary viral pneumonias accounted for about 20% of deaths (Kuiken and Taubenberger, 2008). However, the mechanism of lung injury in these cases is not clear, since epithelial apoptosis alone is not sufficient to induce lung leak (Mura et al., 2010).
To date however, a possible effect of influenza on the lung endothelium has been largely overlooked (Teijaro et al., 2011). In vivo, epithelial and endothelial infection, platelet adhesion, and other factors such as systemic cytokines and the release of leukocyte granules may synergize to induce lung injury. To date, however, agents for human influenza targeting the lung endothelium have not been described.
In addition to viral pneumonia, in the 1957 pandemic the remainder of deaths (75%) occurred as a consequence of bacterial superinfection. The classical clinical vignette is of a patient who initially improves after the onset of influenza, only to dramatically deteriorate as early as 2-3 days later (Peltola and McCullers, 2004; Mohan et al., 2005) from bacterial superinfection with Gram-positive organisms like Staphylococcus aureus. Autopsy data from the 1918 flu epidemic and data from animal models led to the commonly held hypothesis that the virus caused immunosuppression leading to diminished clearance of bacteria (Speshock et al., 2007). However, antibiotics were not available in 1918 and are not typically used in animal models. Indeed, in the 1957 flu pandemic when antibiotics were readily available, most autopsy lung cultures were negative (Louria et al., 1959; Oseasohn et al., 1959). Thus, the almost universal administration of empiric broad-spectrum antibiotics (McGeer et al., 2007) to patients with severe flu (due to diagnostic uncertainty) makes bacterial replication per se unlikely as the cause of acute lung injury. In support of this notion, in a mouse model of flu and S. aureus superinfection, death could not be explained by unrestrained bacterial growth, nor could it be attenuated by depletion of leukocytes (i.e. it was not leukocyte-mediated) (Iverson et al., 2011).
The US Centers for Disease Control and Prevention (CDC) recommend antiviral medications with activity against influenza viruses as important adjunct to influenza vaccines in the control of influenza (see world wide web at cdc.gov/flu/professionals/antivirals/summary-clinicians.htm). Antiviral medications are used in the treatment, as well as prevention, of influenza. FDA-approved antiviral medications include oseltamivir (Tamiflu®) and zanamivir (Relenza®). Clinical benefit is greatest when antivirals are administered early, especially within 48 hours of illness onset and has declining efficacy when given delayed.
Angiopoietins (Ang) 1-4 have all been shown to bind to and activate Tie2 receptor tyrosine kinase activity to differing extents. All the Angs are characterized structurally by an N-terminal super clustering domain (SCD) followed by a coiled-coil domain (CCD) and a C-terminal fibrinogen-like domain (FLD) (Ward and Dumont 2002; Tsigkos et al. 2003). Functional studies have highlighted a role for the SCD and CCD in forming high order homotypic multimers of Ang (Procopio et al. 1999). The specific nature of these multimers is variable and is unique to each Ang family member. Binding specificity of the Angs for the Tie2 receptor has been ascribed to the FLD (Tisgkos et al. 2003; Procopio et al. 1999). Taken together, it is the unique structural attributes of each Ang family member that promotes binding and differential clustering of Tie2. The pleiotropic physiological effects of Angs 1-4 are thought to at least in part be mediated by appropriate and specific clustering of the receptor. For instance, mice engineered to overexpress the CCD of Ang 1, capable of multimerizing with endogenous Ang1 produced in the same cell, caused improper patterning of the coronary vessels (Ward et al. 2004). Furthermore, chimeric forms of Ang 1 engineered to contain the C-terminal FLD and one of several different CCDs differed in their ability to activate the Tie2 receptor (Cho et al. 2004a; Cho et al. 2004b).
The present inventors previously designed peptide mimetics of Angiopoietin that bind to Tie2 and when configured as a dimer or tetramer (the tetramer is known as Vasculotide) result in the clustering of the receptor and its activation (Van Slyke et al. 2009, David et al. 2011, and Kumpers et al. 2011; WO2008/049227).
Activating Tie2 through the tetramerization of high affinity Tie2 binding peptides using the biotin/avidin model (Van Slyke et al. 2009) has established the use of the peptide as an agonist to the Tie2 receptor to promote angiogenesis for applications in diabetic wound healing and other cardiovascular indications. Vasculotide has also been shown to protect against vascular leakage. Studies examining the impact of VT on in vitro endothelium permeability as well as in endotoxemia- and polymicrobial-induced sepsis demonstrated that VT was able to prevent and/or reverse endothelial permeability induced by these treatments. Moreover VT was able to prevent the breakdown of EC:EC interactions in vitro further illustrating its ability to inhibit vascular permeability (David et al. 2011).